Non-alcoholic fatty liver disease (NAFLD) is becoming an increasingly prevalent chronic liver disease all over the world. The present study was undertaken to explore the synergistic effects of sea cucumber saponins (SCS) and eicosapentaenoic acid-enriched phospholipids (EPA-PL) at ratios of 0.5 : 0.5 and 1 : 1 on NAFLD and demonstrate possible protective mechanisms. It was found that the combination of EPA-PL and SCS at half dose exhibited better effects than EPA-PL or SCS alone and the combination of EPA-PL and SCS at full dose in alleviating orotic acid (OA)-induced symptoms including growth parameters, serum parameters and liver function. Further evaluation of the mechanism illustrated that EPA-PL and SCS combination at the ratio of 0.5 : 0.5 could markedly reduce the mRNA expressions of fatty acid synthase, acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase and malic enzyme genes and significantly increase expression of genes relevant to fatty acid β-oxidation including peroxisome proliferator-activated receptor and its target genes , suggesting that the protection of the EPA-PL and SCS combination at the ratio of 0.5 : 0.5 against OA-induced NAFLD might be mainly via lipogenesis inhibition and β-oxidation enhancement in the liver. The synergistic effects of EPA-PL and SCS make it possible to reduce the doses of EPA-PL or SCS to avoid side effects, which is of value for the development of dietary supplements or functional foods for preventing or treating NAFLD. NAFLD has been associated with insulin resistance and metabolic syndrome . Orotic Sea cucumber is a conventional tonic in China, and it contains a wide variety of bioactive compounds such as polysaccharides, saponins and phospholipids . Growingn-3 polyunsaturated fatty acids (PUFAs) could increase lipid peroxidation (LPO) [However, the raw materials of EPA-PL are scarce and EPA is readily oxidized . More imon (LPO) . In addion (LPO) ,14. Theron (LPO) ,16.Theoretically, a combination of EPA-PL and SCS could show synergistic effect in lipid metabolism. However, there are no reports about synergistic effects of EPA-PL and SCS on OA-induced NAFLD. To evaluate the synergistic effects of EPA-PL and SCS on lipid metabolism in dietary OA-induced NAFLD rats, we measured the lipid content in the serum and liver, and the expression of crucial genes involved in hepatic fatty acid synthesis and β-oxidation.2.2.1.Pearsonothuria graeffei by a previous procedure [P. graeffei and was then extracted four times with refluxing ethanol. The combined extracts were evaporated in vacuo and further partitioned between water and chloroform. The water layer was extracted with n-butanol and the organic layer was evaporated in vacuo to yield n-butanol extracts. Samples were applied on an HP20 resin column, and eluted with water, 80% ethanol and 100% ethanol in sequence. The fraction eluted with 80% ethanol was collected and evaporated to obtain SCS. The purity of the yielded SCS was 80.4% and the main ingredients were echinoside A and holothurin A according to our previous study [The saponins were isolated from the sea cucumber rocedure . The powus study .Cucumaria frondosa was purchased in the Nanshan aquatic market of Qingdao. The body walls of C. frondosas were ground into powder after vacuum freeze-drying. Then the powder was extracted by 20 times of the chloroform solution–methanol solution  overnight. The extracted solution was mixed with one-fourth of water after filtration. The mixture was placed into a separating funnel and maintained for 24 h and then the chloroform layer was collected and evaporated to dryness under vacuum, containing the total lipids. Then EPA-PL was separated from neutral lipids and glycolipids by silica-gel column chromatography using chloroform, acetone, chloroform–methanol , chloroform–methanol  and methanol sequentially as eluents. The chloroform–methanol  eluent and methanol eluent were collected and EPA-PL was obtained after the removal of organic solvent under vacuum. The purity of EPA-PL was confirmed to be 84.1% according to the method of Ames [−1 and held at 240°C for 15 min. Nitrogen was used as the carrier gas at a flow rate of 1.2 ml min−1. The main fatty acid composition of EPA-PL is shown in The EPA-PL was prepared according to a previous method . The fre of Ames . The fat2.2.ad libitum. After a one-week adaptation period, the animals were divided into six groups consisting of seven rats each group. Rats were assigned to the following groups: (1) the normal control group (Con); (2) the orotic acid model group (OA); (3) the EPA-PL group; (4) the SCS group; (5) and (6) the combination of EPA-PL and SCS at half dose  and full dose  groups. The feed of each group was prepared on the basis of AIN93-M basic formula. The Con was supplied with an ordinary diet and the diets of the other five groups were prepared by supplementation of 1% orotic acid to the basal diet at the expense of corn starch as previously used. The EPA-PL group and SCS group were fed with 1% EPA-PL and 0.028% SCS, respectively. The ingredients of the six experimental diets are summarized in All aspects of the experiment were conducted according to the guidelines provided by the ethical committee of experimental animal care at Ocean University of China . SD rats , weighing 240 ± 10 g at the beginning of the experiments, were acclimatized under a 12 L : 12 D cycle at 23°C and provided with food and water 2.3.Hepatic lipids were extracted and purified according to the method of Folch  and then2.4.Serum TG, TC, free fatty acid (FFA) and glucose levels, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured using enzymatic reagent kits according to the manufacturer's instructions  .2.5.Real time-PCR (RT-PCR) was used to measure mRNA levels of related genes according to previous studies ,24. Tota2.6.post hoc test. The level of significance chosen was p < 0.05.All results were expressed as mean ± s.e.m. (indicated by error bars). Statistical analyses were performed with SPSS18.0. Differences between the means were tested by one-way ANOVA, and all detected significant differences were further evaluated by Tukey's 3.3.1.p < 0.05) and 43.0% (p < 0.01), respectively, while there was no significant difference in perirenal adipose tissue weight. This was consistent with the previous studies, which also showed the significant fatty liver of rats in the model of OA-induced NAFLD [As described in % p < 0.0, respectn-3 PUFA-rich fish oil exhibited a positive effect on lowering the liver weight in high-cholesterol diet-induced NAFLD [Interestingly, only the half dose combination of EPA-PL and SCS could increase the BW after treatment for 10 days, reaching a weight close to the Con group. The combination of EPA-PL and SCS at full dose noticeably downregulated the OA-induced liver weight by 17%, which showed excellent improvement. Additionally, EPA-PL and EPA-PL : SCS at the ratio of 0.5 : 0.5 also decreased liver weight to some degree, while there was no statistical difference. Importantly, the EPA-PL and the combination of EPA-PL and SCS at half dose significantly reduced the OA-induced weight increase in epididymal and perirenal tissue. Previous studies showed that ed NAFLD . Overall3.2.p < 0.001 and p < 0.05, respectively). A previous study also indicated that OA diet supplementation for 30 days could reduce the levels of serum TG and TC without significance [NAFLD may be partly related to an increased risk of underlying metabolic abnormalities, such as glucose intolerance and dyslipidaemia . We deteEPA-PL treatment for 10 days could significantly increase both serum TG and TC levels in NAFLD rats fed with the OA diet. However, EPA-PL showed a positive effect on reducing serum TC and TG in diet-induced-obese mice ,7. The a3.3.The liver plays a central role in lipid metabolism, which likely contributes to the onset and progression of chronic disease . Orotic et al. [C. frondosas had protective effects against NAFLD [When the OA-induced rats were treated with EPA-PL alone, and the combination of EPA-PL and SCS at half or full dose for 10 days, the hepatic TG and TC contents were significantly reduced by different degrees. In another study, Liu et al.  also foust NAFLD . HoweverThe effects of samples on lowering hepatic lipids levels might be attributed to enhancing cholesterol excretion and improving the hepatic parameters of AST and ALT. Therefore, the AST and ALT levels were evaluated in the next experiment.3.4.p < 0.01), respectively, compared with the control group, indicating that rats fed with OA diet displayed severe NAFLD with liver injury. Those results were in agreement with our previous studies [Serum AST and ALT levels as biochemical markers for liver health are commonly measured clinically.  studies ,34.Figua, electronic supplementary material, data S4). EPA-PL and the half dose combination of EPA-PL and SCS had the same effect on reducing AST content to some degree, but without significance in comparison with OA group . Interestingly, EPA-PL and the combinations of EPA-PL and SCS at half or full dose all could dramatically decrease ALT activity in NAFLD rats . Several studies have suggested that EPA is effective in reducing the serum AST and ALT activities [After the treatment with samples for 10 days, only the combination of EPA-PL and SCS at full dose could significantly decrease AST activity in OA-induced rats  regulates the transcription of genes in lipid metabolism including fatty acid and TG metabolism [The sterol-regulatory element-binding proteins (SREBPs) are central in the regulation of lipid biosynthetic pathways . SREBP-1tabolism . The lipp < 0.05) and of the relevant genes including FAS, ACC, G6PDH and ME (a). Meanwhile, SCS showed an improvement similar to the combination of EPA-PL and SCS at half dose although there was no significant difference in comparison with the OA group. Importantly, the combination of EPA-PL and SCS at half dose could markedly reduce the expression levels of FAS, ACC, G6PDH and ME mRNA, which were very close to those of the control group (b–e). Moreover, the combination of EPA-PL and SCS at full dose significantly decreased the FAS, ACC and ME mRNA levels rather than that of G6PDH. The combination of EPA-PL and SCS inhibited the mRNA expression of ACC, FAS, ME and G6PDH by directly reducing SREBP-1c expression, which could result in the inhibition of hepatic endogenous fatty acid synthesis. Treatment with EPA-PL alone showed obvious downregulation only in G6PDH and ME mRNA levels, indicating that EPA-PL might decrease the liver lipids mainly through inhibiting the support of energy in fatty acid synthesis [Results showed that OA administration markedly increased the mRNA expression of hepatic SREBP-1c , which was in agreement with previous studies [n-3 PUFAs could enhance fatty acid β-oxidation. However, the present study revealed that the SCS showed a converse effect on the mRNA expression of PPARα, CPT1 and CPT2, and it could significantly increase the ACOX1 mRNA level by 65%. These results were different from the previous study, which found the PPARα, CPT1, CPT2 and ACOX1 expression were significantly upregulated by 0.05% SCS treatment [OA remarkably reduced the oxidation capacity, particularly downregulating the mRNA levels of CPT2 and ACOX1 ( studies , further studies . In addi studies ,42, whicreatment . The difOverall, the combination of EPA-PL and SCS at half dose could significantly protect rats against OA-induced NAFLD mainly through inhibiting fatty acid synthesis and enhancing fatty acid β-oxidation .Figure 4.In conclusion, the present study evaluated the synergistic effect of EPA-PL and SCS on OA-induced NAFLD rats by determining growth parameters, serum parameters and liver function. Results showed that dietary supplementation of EPA-PL and SCS at half dose could markedly alleviate OA-induced symptoms. Further elucidation showed that the protective mechanism of the combination of EPA-PL and SCS at half dose against OA-induced NAFLD was mainly by inhibiting lipogenesis and enhancing fatty acid β-oxidation. The synergistic effects of EPA-PL and SCS make it possible to reduce the doses of EPA-PL or SCS to avoid side effects, which is of value for the development of dietary supplements or functional foods for preventing or treating NAFLD.


This randomized controlled clinical trial aimed to evaluate the effect of the casein phosphopeptide-amorphous calcium phosphate fluoride (CPP-ACPF) and photobiomodulation (PBM) in the treatment of dentin hypersensitivity (DH), and the impact of this on the health-related quality of life (HRQL).st, 2nd and 3rd treatment sessions and one-month follow-up. The HRQL was recorded in the DH experience questionnaire (DHEQ).Eighty teeth with DH were randomized into four groups and received three treatment sessions: PLACEBO = placebo + LASER application mimicking; CPP-ACPF = CPP-ACPF + LASER application mimicking; PBM = placebo + LASER active application; CPP-ACPF+PBM = CPP-ACPF + LASER active application. Tactile (exploratory probe) and evaporative (triple syringe) stimuli were used to measure DH and were recorded with the aid of a visual analogue scale (VAS) after the 1The intragroup comparison showed a significant reduction in DH (p < 0.05) with both stimuli after one-month follow-up. The intergroup comparison with the evaporative stimulus showed that CPP-ACPF+PBM significantly reduced DH when compared to the rest of treatments, after one-month follow-up. CPP-ACPF+PBM group statistically differed from the other treatment groups in the DHEQ evaluation after one-month follow-up.After one-month follow-up, the association of CPP-ACPF with PBM was effective in the reduction of DH and promoted a positive impact on the HRQL of the participants of this study. Dentin hypersensitivity (DH) is characterized by an acute and short-term pain, arising from vital dentin exposed to the oral medium, in response to thermal, evaporative, tactile, osmotic, or chemical stimulation ,2. BrännDH prevalence ranges from 8% to 98% according to the evaluated population –7. DH deTreatments that obliterate the dentinal tubules are ideal to reduce DH, however, their effectiveness will depend on their resistance to the various challenges of the buccal environment . RecentlPhotobiomodulation (PBM) involves noninvasive application of red (600–700 nm) and near-infrared light (700–950 nm) , it is iSeveral treatments for DH are available but none are considered the "gold standard," since most desensitizing agents have shown short-term effectiveness about up to two weeks from treatment conclusion ,16, demoCPP-ACPF and PBM association may generate a synergistic effect in DH treatment. This double-blind, parallel, placebo-controlled randomized clinical trial (RCT) evaluated the effect of CPP-ACPF associated with PBM on the permanent teeth of adult patients with DH. The null hypotheses of this study were: H0—There is no difference in the reduction in DH between the CPP-ACPF and PBM association and other types of treatments after one-month follow-up. H01—There is no difference in the improvement of HRQL among participants after treatment with CPP-ACPF associated with PBM and other types of treatment after one-month follow-up.https://clinicaltrials.gov/ct2/show/NCT03750851?cond=CPP-ACPF&rank=1). After being duly informed about the risks and methods of this study, all the participants signed an informed written consent in accordance with the Declaration of Helsinki [The CONSORT  recommendations  and its Helsinki .® program , with a statistical power of 80%, a probability of error α of 5%, and effect size f estimative of 1.35 a total of 08 teeth per arm would be necessary. An inflation using the design effect was also calculated, based on an estimate ρ of 0.50, and assuming a 20% sample loss a total of 80 teeth were required. Participants were allocated into four groups according to the treatment applied as follows: PLACEBO: water-based placebo gel ; CPP-ACPF: MI Paste Plus ; PBM: LASER—; and CPP-ACPF+PBM: MI Paste Plus + LASER.The data obtained in a pilot study were introduced into the G PowerAdults between 18 to 50 years of age, who reported dental sensitivity, were evaluated at the Federal University of Pará (UFPA), Brazil. Those who met the following inclusion criteria were recruited: at least two sensitive teeth with a response ≥ 4 on the 10 cm length visual analogue scale (VAS) (0—no pain and 10—unbearable pain) after tactile and evaporative stimulation and/or the presence of a non-carious lesion up to 2 mm deep , and/or One of the research collaborators performed a block randomization using a numerical draw which allowed each participant to be allocated into one of the four groups under different arrangements . The allocation concealment was maintained since the numerical draw was performed using numbered and coded papers. The code for each group was unknown by all: participants, principal investigator, clinical operator, and evaluator. In this double-blind study, participants were unaware of the desensitizing treatment received because both pastes were contained in similar recipients hindering visual identification and the texture, color and odor of the placebo was similar to MI Paste Plus. The evaluator was not aware of the group to which the participant belonged.All the participants received a toothbrush with soft bristles  and a dentifrice without a desensitizing action  with oral hygiene instructions. Three desensitizing treatment sessions were performed with a 24-hour interval between each session.The PLACEBO group received a water-based base paste application with a microbrush  on the cervical vestibular surfaces of each tooth for five minutes that was later rubbed for 20 seconds with a rubber cup  coupled to a low speed handpiece . Followed by PBM simulation, that was carried out by positioning the LASER tip at two points of each tooth, one at the center of the cervical region and another one in the middle third of the crown without light emission. The sound of the equipment was mimicked by an applicative on the cell-phone .2 at each point) for 16 seconds. In the CPP-ACPF+PBM group, MI Paste Plus and PBM were applied following the CPP-ACPF and PBM protocols.CPP-ACPF group participants received the MI Paste Plus application following the PLACEBO group protocol, the PBM was simulated in the same way. For the PBM group, the placebo paste was applied as previously described and the LASER was applied using an infrared light spectrum with a wavelength of 808 nm, positioning its tip at the previously described points , perpendicularly to the buccal surface of the tooth (0.5 cm distance), the adjacent teeth were protected with cotton rolls ,24. DH wThe participants’ self-reported evaluation was performed by giving them a questionnaire before the start and after one-month follow-up to determine the treatment impact on their HRQL ,26. Its VAS values were analyzed with the SPSS program . A mixed-design ANOVA test was used to determine whether some of the treatments (between—subjects factor) were more effective for DH treatment throughout time (within—subject factor). Accounting for treatment effects at the different evaluation times were analyzed by a Tukey post-hoc test, tests of multiple comparisons were corrected using a Bonferroni adjustment. A 5% significance level was adopted for all the analyzes. Tooth level analyses were performed wherein each tooth individually contributed to the analyses. Hence this cluster data the subject was included as a random effect.DHEQ data was analyzed by the Wilcoxon and Mann-Whitney tests. The impact of the effectiveness of DH treatment on the HRQL improvement was assessed by simple logistic regression. The difference between the final DHEQ and the initial DHEQ was transformed into binary data. Considering values ≥ 0 as worsening and < 0 values as an improvement in HRQL. The VAS final mean with an evaporative stimulus > 4 was considered as ineffective and ≤ 3 as effective in the DH treatment.st June 2018 and the 20th of October 2018, 40 participants were evaluated and 24 were randomized, treated, and accompanied according to nd session, 21 participants (87.5%) with 80 teeth completed all phases of the study.Between the 1Demographic characteristics of the 21 participants who completed the study are shown in nd (p = 0.006), 3rd session (p = 0.004), and One-month follow-up (p < 0.001). The CPP-ACPF and CPP-ACPF+PBM groups showed a significant reduction in DH after all sessions and one-month follow-up (p < 0.001).A mixed ANOVA test demonstrated that there was a significant difference in the different evaluation times F = 44,661; (p < 0.001). While time and treatment interaction did not reflect a significant difference, F = 1,567; (p = 0.123). The mean and standard deviation of the VAS scores obtained with the tactile stimulus are described in st session, the CPP-ACPF+PBM group showed a higher and significant DH reduction when compared to the PLACEBO group (p = 0.053) and to the PBM group (p = 0.005). In the same period, the CPP-ACPF group showed a significant reduction in DH in relation to the PBM group (p = 0.05). After the 3rd session, the CPP-ACPF+PBM group DH significantly reduced when compared to the PLACEBO (p = 0.009) and the CPP-ACPF groups (p = 0.043). After one-month follow-up, the CPP-ACPF+PBM group had a higher reduction in DH when compared to PLACEBO and PBM groups (p < 0.05) but did not differ from the CPP-ACPF group (p = 0.134).The intergroup comparison showed that the DH baseline mean was similar among all groups (p = 1.00). After the 1rd session (p = 0.024) and one-month follow-up (p = 0.004). The CPP-ACPF group showed a significant reduction after the 2nd (p = 0.010), 3rd session, and one-month follow-up, (p < 0.001). The PBM and CPP-ACPF+PBM groups presented a significant reduction in DH after the 1st (p = 0.002), 2nd, 3rd session, and one-month follow-up, (p < 0.001).A mixed ANOVA test demonstrated that there was a significant difference among the evaluation times, F = 43.53; (p < 0.001), and in the time and treatment interaction, F = 2.68; (p = 0.003). The mean and standard deviation of the VAS scores obtained with the evaporative stimulus are described in st and 2nd session among all the groups (p > 0.05). After the 3rd session, the CPP-ACPF+PBM group had a significant reduction in DH when compared to the PLACEBO group (p = 0.050) but did not differ significantly from the other groups (p > 0.05). After one-month follow-up, the CPP-ACPF+PBM group showed a significant reduction in DH when compared to the PLACEBO (p = 0.007), CPP-ACPF (p = 0.040), and PBM (p = 0.050) groups.The intergroup comparison showed a similarity in the VAS means obtained at the baseline evaluation and after the 1The Wilcoxon test showed a significant difference in the overall analysis between pre-treatment versus post-treatment HRQL (p = 0.031). The intragroup analysis showed that the DHEQ median differences before and after the desensitizing treatment were not different in the PLACEBO (p = 0.893), CPP-ACPF (p = 0.345), and PBM (p = 0.500) groups. The CPP-ACPF+PBM group showed a significant improvement in the HRQL (p = 0.028). The intergroup analysis showed that the DHEQ medians at baseline were similar for all the groups (p > 0.05). After one-month follow-up, the CPP-ACPF+PBM group had lower DHEQ values, thus, a significant improvement in the HRQL when compared to the PLACEBO, Z = 2,739 (p = 0.006), CPP-ACPF and PBM groups, Z = 2.37 (p = 0.018) .Of the 21 evaluated participants, 15 experienced an improvement in their HRQL. The DH treatment was effective in 12 participants. A simple logistic regression analysis showed a relationship of dependence between the HRQL improvement and the desensitizing treatment effectiveness after one-month follow-up. A desensitizing effective treatment has a chance of 91.67%, an OR = 13.7500; and a 95% CI = 1.21–156.66, (p = 0.0347) of improving the HRQL.DH is related to repeated episodes of acute pain and a short duration, characterizing a chronic pain process . The ideThe demographic analysis showed that most of the participants were female, agreeing with the previous studies (62% -72.2%) –30. ThisThe positive interaction between the time and treatment factors demonstrated the impact of the number of applications to achieve a better efficacy. After one-month follow-up, all treatments significantly reduced DH, including the placebo group, revealing effectiveness. The positive response to the placebo treatment may be related to the impact of psychosomatic phenomena, spontaneous improvement, positive responses by courtesy and experimental subordination . The psyThe PLACEBO and the CPP-ACPF and PBM groups similar results could be explained by the fact that a positive professional approach has been related to DH reduction by increasing the participants trust and inhibiting the pain controlled by the central nerve system . The plaCasein phosphopeptide (CPP) is a casein-derived phosphoprotein whose phosphoserine sequences have the ability to bind and stabilize the soluble amorphous calcium phosphate (ACP). In the presence of saliva, ACP dissociates into calcium and phosphate which bind to the tooth surface and provide a reservoir of large amounts of calcium and phosphate ions, maintaining a relative state of dental mineral supersaturation and favoring remineralization by precipitating in the form of hydroxyapatite (HA), occluding the dentinal tubules ,37. FluoIn the PBM group with the evaporative stimulus, a significant reduction in DH was evidenced after the 1st session, showing the immediate analgesic action produced by the PBM. DH treatment with low energy dose emissions stimulate circulation, suppress neuronal transmission at the dentin-pulp interface when inhibiting the depolarization by formation of temporal bends’ in the C fibers axons, blocking 30% of neuronal activity after 10–20 minutes of application . PBM’s aThe adhesion of the CPP-ACPF complexes to the dentinal surface relies on the higher ability of the CPP-ACP compounds to dissolve in aqueous solutions, such a saliva, which generates a diffusion gradient , that coin vitro study [Few studies evaluate desensitizing products based on calcium and phosphate . No studro study  the syneEven though the participants baseline characteristics demonstrated a sample homogeneity, and the non-carious cervical lesions (NCCL) depth was up to 2mm aiming to control this factor, the different lesions depths of the sensitive NCCL could be considered a limitation of the current study since deeper lesions could turn the DH regression a more complex process . Even ifThe DHEQ presents excellent psychometric properties with an adequate reliability  and validity (r = 0.92), properly assessing functional and personal changes in patients with DH ,26. AfteThe DHEQ and VAS results confirmed the impact of the desensitizing treatments on DH and the HRQL of the participants of this investigation. CPP-ACPF with a PBM synergistic effect was effective in DH treatment and promoted a positive impact on the HRQL of participants. Further studies should be performed to evaluate the protocols of combination therapies with longer treatment periods.Within the limitations of this RCT, it was concluded that the association of CPP-ACPF with PBM was effective in reducing DH after one-month follow-up and promoted a positive impact on the HRQL of the participants of this study.S1 Checklist(DOCX)Click here for additional data file.S1 Protocol(DOCX)Click here for additional data file.


The bone marrow (BM) provides a preferential survival environment for the long-term maintenance of antigen-experienced adaptive immune cells. After the contact with antigens, effector/memory T cells and plasma cell precursors migrate to the BM, in which they can survive within survival niches in an antigen-independent manner. Despite this, the phenotype of adaptive immune cells changes with aging, and BM niches themselves are affected, leading to impaired long-term maintenance of immunological memory in the elderly as a result. Oxidative stress, age-related inflammation (inflammaging), and cellular senescence appear to play a major role in this process. This review will summarize the age-related changes in T and B cell phenotype, and in the BM niches, discussing the possibility that the accumulation of highly differentiated, senescent-like T cells in the BM during aging may cause inflammation in the BM and promote oxidative stress and senescence. In addition, senescent-like T cells may compete for space with other immune cells within the marrow, partially excluding effector/memory T cells and long-lived plasma cells from the niches. The bone marrow (BM) is renowned for hematopoiesis, the formation of the cellular components that comprise our blood, but it has also been described as the preferred site for the maintenance and survival of long-living immune cells ,2. Over A key role in memory T cell homing and survival has been attributed to the BM. Resting memory T cells specific for systemic pathogens such as tetanus, rubella, mumps, and measles can be found in the human BM . After a+ and CD8+ T cells . I. I+ and  T cells . In the  T cells , and intescribed . The maind IL-15 . While a CD132) . A schemignaling . Indeed,by IL-15 . Whethern the BM ,30,31. I . A scherophages .+ T cells relocate to the BM during the contraction phase of a reaction, and after 120 days, antigen experienced cells were only detectable in the BM [in situ labeling of sheep BM cells, which subsequently reached the spleen and other secondary lymphoid organs, supports the theory that the BM is a temporary stopping point for recirculating memory T cells, which find in the BM a proper environment to be maintained by homeostatic proliferation [+ and CD45.2+ antigen specific memory CD8+ T cells were detectable in the BM of each mouse [Two major theories exist on the long-term maintenance of memory cells in the BM: the quiescent niche and the self-renewal niche ,37. Evidn the BM . Alternan the BM . The in feration ,37,39. Ach mouse . In addiThe age-related decline in the functionality of the immune system, which leads to impaired immune responses in the elderly, is known as immunosenescence ,42. ThisOnce the immune system has encountered a pathogen, it can develop immunological memory, allowing a faster and more effective response should a second exposure occur . B lymphet al. reported changes with age within the different B cell subsets, as well as shifts in the diversity and clonotypic composition of the antigen recognition repertoire [Cancro pertoire . The autpertoire . A decrepertoire . Within pertoire . B cellspertoire . The regpertoire . A recenpertoire . Poor repertoire ,52. Addipertoire , as wellpertoire . Considepertoire .+ and CD8+ T cells are affected by aging, age-related changes are more evident in CD8+ T cells. In the elderly, high frequency of CD8+ T cells lack co-stimulatory molecules important for T cell activation and proliferation, such as CD28 [+ and CD4+ T cells gain the expression of CD57 [+CD28− T cells are characterized by short telomere length, reduced antigen-induced proliferation, and enhanced production of pro-inflammatory cytokines [One of the leading causes of immunosenescence and aging itself is the involution of the thymus . After b as CD28 . In addi of CD57 ,65. CD8+ytokines ,66–68.+CD28− T cells have often been considered a population of senescent T cells and are believed to contribute to a low grade age-associated inflammatory background (‘inflammaging’) [+ T cells has also been associated with a reduced immune response to pathogens and vaccines in old age [+ and CD8+ naive T cells declines while effector memory T cells increase in the BM in old age [+ and CD8+ T cells in paired PB and BM samples, naive T cells were reduced while effector/memory T cells accumulated in the BM, underlining the importance of BM in the maintenance of immunological memory [+CD28− T cells increase during aging in the BM [+ and CD8+ T cells, T cells which simultaneously produce multiple pro-inflammatory cytokines such as interferon γ (IFNγ) and as tumor necrosis factor (TNF) [For these reasons, CD8maging’) . The acc old age . Similar old age ,71,72. Wl memory . In addior (TNF) , residesor (TNF) .The function of cytotoxic T cells in BM is not completely understood. However, it is known that these cells play an important role within the BM environment because of their IFNγ production. IFNγ promotes the release of hematopoietic cytokines, including IL-6, from BM mesenchymal cells in the HSC niche, which at low concentrations stimulates hematopoiesis and induces myeloid differentiation . DespiteIn summary, although life-long maintenance of polyfunctional effector/memory T cells and T cell cytotoxicity in the BM seem to be unaffected by age, immunosenescence does take place with advancing age. Indeed, the phenotype of effector/memory T cells that reside in BM niches in old age is very different to the state in younger individuals, in regard to the expression of co-stimulatory molecules (CD28) and cytokine receptors  .As the immune system ages, the production of pro-inflammatory molecules such as TNF, and IL- 6 increases in the serum over time ,77. Thiset al., in which BM stromal cells derived from old donors produced less IL-7 than stromal elements derived from young BM [+ T cells in humans and mice [We recently described the effects that aging has on the production of some survival factors for effector/memory T cells and long-lived plasma cells in the human BM . In partyoung BM . IL-15, young BM ,23. Despyoung BM . In addiyoung BM , was notand mice ,23. Despin vitro [+CD28− T cells could be prevented by IL-15, suggesting that this cytokine may play a role in the survival and the age-related accumulation of CD8+CD28− T cells [+CD28− T cells increased during aging in the BM, we theorize that IL-15 may strongly support the survival of this subset of highly differentiated T cells [+CD28– T cells in the BM; therefore, BM niches involved in the long-term maintenance of memory T cells, appear to progressively become pro-inflammatory in old age, with CD8+CD28− T cells supporting this process. Indeed, after their recruitment to the BM, CD8+CD28− T cells secrete IFNγ and TNF, which may act on BM cells and promote the production of IL-15 and IL-6, promoting the attraction of new CD8+CD28− T cells as a result. Therefore we suggest that a vicious cycle of inflammation may take place in the aged BM, compromising the maintenance of immunological memory as a result.After having described that the levels of both IFNγ and TNF increase in the BM in old age, we also observed that IFNγ in particular could induce the production of IL-15 and IL-6 in myeloid cell types in vitro . It has  T cells . As we oAnother factor which may contribute to the age-related dysfunctions in the maintenance of immunological memory in the BM is CMV. CMV is a highly prevalent, persistent β-herpes virus establishing a life-long infection, during which functional changes in the phenotype of T cells take place ,84. With+ T cells, and a high fraction of the CD8+ T cell compartment is required to fight the virus. It is believed that 10% or more of circulating T cells are required to recognize CMV antigens and control the viral proliferation [+ T cells accumulate throughout the body, persisting for a long time, even when the infection becomes latent [+ T cells have been shown to accumulate in the BM of CMV+ individuals [+ T cells with reduced levels of CD28, and expressing senescence markers such as killer cell lectin like receptor G1 (KLRG1) dramatically increases in comparison to their seronegative counterparts [+ T cells and IL-15-producing cells have been reported in the presence of CMV [+ T cells preferentially respond to IL-15, due to their high expression of CD122 (IL-2/IL-15Rβ) and reduced levels of IL-7Rα, the increased levels of IL-15 [+CD28+ memory T cells is therefore impaired. CMV infection is associated with and may contribute to inflammaging’. Highly differentiated, antigen-experienced CD8+CD28− T cells with a pro- inflammatory phenotype accumulate with CMV in the PB and they may also migrate to BM when they circulate [+ persons [+ compared with CMV-individuals [The immune response to CMV is primarily controlled by cytotoxic CD8feration . Even ins latent ,89. Amonividuals . In seroterparts . This octerparts , 91. In e of CMV . As lateof IL-15  may suppirculate ,93. CMV irculate . A negatirculate ,98. Low  persons . 5 yearsividuals . TherefoIn vitro, PBMC stimulation with IFNγ leads to increased ROS levels in blood-derived (CD11chi CD14+) myeloid cell types [in vivo experiments; however, would need to be conducted to confirm these results. These data all provide further evidences about the important role that the BM plays in supporting adaptive immune cells against systemic pathogens.In addition to inflammation, reactive oxygen species (ROS) may also interfere with the survival of adaptive immune cells in the BM. This hypothesis is supported by the observation that infiltrating leukocytes such as macrophages may constantly produce oxygen radicals, which play an important role in killing pathogens, eventually contributing to structural damages of tissues . Aging ill types . In parall types . Therefoll types . In addill types . These sAge-related inflammation may also be due to cellular senescence ,105. SenIn the elderly, oxidative stress, age-related inflammation (inflammaging), and cellular senescence alter the BM environment, possibly changing the structure of BM niches themselves . In addiDespite this, vitamin C and NAC are very safe; therefore offering promising strategies to boost adaptive immunity.Other possible interventions may be beneficial to combat the impact of aging on BM. In particular, physical activity (cycling) has been shown to increase IL-7 and decrease IL-6 serum levels, improve thymus aging, and reduce Th17 polarization and B regulatory cell frequency . In addi


The Breastfeeding Self-Efficacy Scale-Short Form (BSES-SF) is a widely used instrument that measures breastfeeding self-efficacy. This study aimed to examine the reliability and validity of the Persian version of BSES-SF in Iranian mothers.2/df = 4.42; CFI = 0.96; NFI = 0.95; IFI = 0.96; RMSEA = 0.095 and SRMR = 0.054). The divergent validity of the BSES-SF was proved via a significant negative correlation with scores of the Edinburgh Postnatal Depression Scale . In sum, the Persian version of the BSES-SF is a reliable and valid instrument for measuring breastfeeding self-efficacy in Iranian mothers.The English version of BSES-SF was translated into Persian using the standard forward–backward translation procedure. No changes  were made to the BSES-SF items. The mean BSES-SF total score was 50.80 ± 8.91. The Cronbach’s alpha coefficient for internal consistency for the BSES-SF was 0.910. The confirmatory factor analysis results provided evidence for unidimensionality of the scale . Breastfeeding is the optimal method of feeding and nurturing infants . It has The Breastfeeding Self-Efficacy Scale (BSES) was developed by Dennis and Faux in 1999 to assess breast-feeding confidence . This scDue to the declining breastfeeding rates in Iran, it was considered essential to validate a Persian version of the BSES-SF and use it as one component in a strategy for improving the breastfeeding rate. Therefore, this study aimed to evaluate the reliability and validity of the BSES-SF as a measure of breast-feeding confidence among Iranian mothers.In this cross-sectional study, breastfeeding mothers referring to a health center for neonatal vaccination in Tehran, Iran were invited to participate in the study. We collected data between July and September 2017. The sample size was determined using the rule of thumb suggested in the literature. Norusis  indicateThe English-language version of the BSES-SF was translated into Persian using the standard forward–backward translation procedure. First, items and response choices were translated into Persian independently by two native Persian speakers, both of whom were fluent in English. The two forward translations were then combined into one version by the research team. This forward translation was translated back into English by a professional translator and compared to the original scale. Additional small changes were performed to ensure that the Persian version did not differ from the original English version. No changes  were made to the BSES-SF items.Basic demographic and obstetric characteristics of the mother and infant included: mother’s age, level of education, occupation, duration of the marriage, parity, delivery mode, type of pregnancy, infant age, and infant sex.The BSES-SF is a 14-item self-administered instrument derived from the original 33-item BSES that measures breastfeeding confidence . All iteThe EPDS is a commonly used self-administered instrument that measures postnatal depression . RespondThe PSS-10 is a commonly used self-administered instrument derived from the original 14 item PSS (PSS-14) that measures perceived stress . The PSS2/df), the comparative fit index (CFI), incremental fit index (IFI), the normed fit index (NFI), the root mean square error of approximation (RMSEA), and the standardized root mean square residual (SRMR). Values of χ2/df < 5, CFI, IFI, and NFI > 0.90, and RMSEA and SRMR < 0.08 indicate an adequate fit of the model to the data [To examine the unidimensionality of the BSES-SF, we performed a confirmatory factor analysis using maximum likelihood estimation method. Model fit was evaluated using several goodness-of-fit indices including the Chi-square/degree of freedom  and LISREL 8.80 .The mean maternal age of the mothers was 30.13 years . Of the participants, 29.8% were university-educated, 12.9% were employed, 57.6% were primiparous. CS was reported for 72.7% of the sample (of which 65.3% responded planned CS (Table The BSES-SF items and their mean and standard deviation (SD) are presented in Table The Cronbach’s alpha coefficient for the BSES-SF was 0.910 and was not increased by more than 0.10 if any item was deleted. All corrected item-total correlations were within acceptable range except for Item 5 (“Manage the breastfeeding situation to my satisfaction”). This item had a corrected item-total correlation of 0.270. The mean corrected item-total correlation for the 14 items was 0.624. The mean interitem correlation was 0.428, with values ranging from 0.068 to 0.752. According to the inter-item correlation matrix, Item 1 and Item 5 had low association with other items.2/df = 5.82; CFI = 0.95; NFI = 0.94; IFI = 0.95; RMSEA = 0.113 and SRMR = 0.064). Examination of the modification indices recommended allowing covariance between Item 1 and Item 2 as well as between Item 3 and Item 4 . As shown in Additional file To test the unidimensionality of the BSES-SF, the CFA was carried out. The goodness-of-fit indices indicated that the model did not fit the data well , indicating satisfactory divergent validity. However, contrary to our expectation, there was no significant relationship between BSES-SF and PSS-10 scores .As presented in Table This study examined the reliability and validity of the BSES-SF in a sample of Iranian mothers. The internal consistency of the BSES-SF was proved as both Cronbach’s alpha and corrected item-total correlations were high. Similar results have been found in other studies validating the BSES-SF , 24, 37.Evidence of divergent validity of the BSES-SF was proved by high negative correlation with the EPDS. This result is in line with two validation study conducted in Brazil  and ItalThe mean of BSES-SF was 50.80 (SD = 8.91), which is lower than what was reported in the original BSES-SF and studies conducted in Brazil , Sweden Similar to our study, studies using the BSES-SF in other countries did not find any relationship between breastfeeding self-efficacy and mother’s age, education, or type of delivery , 17, 37.In conclusion, in light of satisfactory reliability and validity, the BSES-SF is a quick and straightforward instrument for the assessment of breastfeeding self-efficacy among Iranian mothers. The findings reported in the current study are following those derived in other validation studies. However, the BSES-SF might be improved if items one and five were removed from the scale. These items had low loadings on the breastfeeding self-efficacy factor, which decreased their internal consistency.There were two limitations in this study that should be noted. First, test–retest reliability was not evaluated. Second, we could not able to follow-up the baby’s feeding and therefore, to examine the predictive validity.Additional file 1: Figure S1. Confirmatory factor analysis of the one-factor model of BSES-SF.


While maintaining anti-tumor efficacy, the levels of body weight loss were significantly lower (p < 0.0001) and the overall degree of neurotoxicity was reduced with Dual-NPs treatment in comparison to the free-drug combination when administered at an equivalent dose of PTX. This study suggests that Dual-NPs present a promising platform for the delivery of the PTX and EVER combination with the potential to reduce severe PTX-induced toxicities and in turn, improve quality of life for patients with BC.A synergistic combination of paclitaxel (PTX) and everolimus (EVER) can allow for lower drug doses, reducing the toxicities associated with PTX, while maintaining therapeutic efficacy. Polymeric nanoparticles (NPs) of high stability provide opportunities to modify the toxicity profile of the drugs by ensuring their delivery to the tumor site at the synergistic ratio while limiting systemic drug exposure and the toxicities that result. The goal of the current study is to evaluate the in vivo fate of human epidermal factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) dual-targeted PTX+EVER-loaded NPs  in an MDA-MB-231-H2N breast cancer (BC) tumor-bearing mouse model. The pharmacokinetic parameters, plasma area under the curve (AUC) and half-life (t Paclitaxel (PTX)-induced toxicities continue to be one of the main challenges associated with the use of this agent. PTX has several dose-limiting adverse effects including neutropenia and neurotoxicity. Additionally, the commercial formulation of PTX (Taxol) has been associated with severe hypersensitivity reactions, mainly caused by the excipients used for drug solubilization  .Over the years, drug-combinations have emerged as one promising avenue for the treatment of cancer. We can take advantage of the synergistic effects of drug-combinations with the administration of doses that are much lower than the maximum tolerated doses (MTDs) of the drugs. This can lead to a reduction in systemic toxicity, while improving or at least maintaining the desired therapeutic effect ,3,4,5.The combination of the chemotherapeutic agent PTX, and the mammalian target of rapamycin (mTOR) inhibitor everolimus (EVER) has previously been shown to have pronounced synergistic effects in breast cancer (BC) cells in vitro . Despite®  is a promising “two-in-one” formulation that includes liposomes that co-encapsulate the combination of cytarabine and daunorubicin at a synergistic molar ratio of 5:1. Vyxeos was approved in 2017 by the FDA for treating patients with high risk acute myeloid leukemia (AML) [Recent advances in cancer nanomedicines provide unique opportunities to modify the toxicity profile of the drugs by increasing their delivery to the target site while limiting systemic drug exposure and the toxicities that result. Vyxeosia (AML) . In a Phia (AML) .b-poly(lactide-co-glycolide) copolymer . The NPs are actively targeted to human epidermal factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR), using HER2-targeted trastuzumab (TmAb) and EGFR-targeted Panitumumab (PmAb) Fab fragments, to deliver the drug combination to BC cells (We have developed and characterized PTX+EVER-loaded NPs composed of poly (ethylene glycol)-BC cells , Table 1Active targeting has been widely utilized to increase the cellular internalization of anticancer therapies into cancer cells, with a goal towards improving therapeutic effects . HER2 ismod/EGFRmod). Additionally, the safety profile of Dual-NPs relative to the free-drug combination was evaluated by examining neurotoxicity and hemotoxicity post administration. This study demonstrates that Dual-NPs have high stability in vivo and, subsequently, result in significant improvements with regard to safety compared to the free-drug combination in the MDA-MB-231-H2N BC tumor-bearing mouse model.The current study builds on the promising results seen with Dual-NPs in vitro and examines the pharmacokinetics and biodistribution profiles of PTX and EVER when administered in the Dual-NPs compared with the free-drug combination in a relevant animal model of BC. Furthermore, the therapeutic effect of PTX and EVER administered in Dual-NPs was evaluated in mice bearing MDA-MB-231-H2N BC tumors which co-express HER2 and EGFR (HER2b-poly(lactide-co-glycolide)  polymeric nanoparticles. TmAb(Fab) and PmAb(Fab) were prepared, purified and conjugated to the surface of PTX+EVER-loaded NPs via amine coupling. The physicochemical characteristics of the Dual-NP are summarized in PTX and EVER were co-encapsulated at the optimal synergistic ratio within poly(ethylene glycol)-max) of PTX and EVER were 7-fold and 9-fold higher for Dual-NPs than for the free drugs, respectively. Furthermore, the half-life (t1/2z) was approximately 3 and 4 times greater for PTX and EVER, respectively, in Dual-NPs than for the free drugs. Analysis of the PTX:EVER molar ratio in the plasma revealed that Dual-NPs maintained the ratio of 1:0.5 for 24 h after administration . The levels of PTX and EVER were measured in the plasma at various timepoints A and thestration B. This mp = 0.27, Evaluation of the biodistribution of PTX+EVER  in tumor-bearing mice was performed at 2, 6, 24 and 48 h after administration. At 24 h post-administration, the Dual-NPs group showed a 2-fold increase in the tumor PTX accumulation compared to the levels detected in the free PTX+EVER group  found in vitro . On the Comparable levels of PTX and EVER were found to accumulate in the hearts and kidneys of mice in the Dual-NPs and free PTX+EVER groups. Differential accumulation of PTX and EVER following administration in Dual-NPs and free PTX+EVER occurred mainly in the spleen and liver. A 2–5 fold and 2–7 fold increase in liver and spleen uptake of PTX was seen in the Dual-NPs group relative to the free-drug combination group, while a 2–4 fold and 2–6 fold increase was noted in liver and spleen uptake of EVER, respectively, for the Dual-NPs group compared with the free-drug combination group. This outcome can be attributed to the clearance of the NPs via the mononuclear phagocyte system (MPS), which is one of the main elimination pathways for NPs .p = 0.001) and day 65 (p = 0.0003) post initiation of treatment in the Dual-NPs group relative to the free-drug combination group (p < 0.0001) (Tumor growth in NOD/SCID mice bearing subcutaneous (s.c.) MDA-MB-231-H2N BC tumors was inhibited following the administration of free PTX+EVER and Dual-NPs. Dual-NPs were associated with enhanced antitumor activity in comparison with the free-drug combination. The extent of tumor growth inhibition was significantly different at day 55 (on group A. In add 0.0001) . As showNeurotoxicity was evaluated using histological examination of the sciatic nerve sections removed two to three days after the sixth weekly injection of saline, Dual-NPs, or free PTX+EVER    analysis was performed using VetScan HM5 v2.2 . Considering the marked increase in Dual-NPs accumulation in the liver and spleen relative to free PTX+EVER, blood samples were also analyzed for biochemistry markers looking mainly for cues of liver and spleen toxicity.For red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit (HCT) and the mean corpuscular cell volume (MCV), values for all groups were within normal levels. Interestingly, the level of neutrophils in mice in all groups was comparable but below the lower normal limit. This outcome was previously seen by Nemzek and colleagues and was attributed to the strain of mice and sampling site used. It was found that in BALB/c mice, when blood is collected from the heart through cardiac puncture, neutrophil levels drop due to the reduced peripheral vascular resistance to the flow of blood cells ,18. OverAlkaline phosphatase (ALP) values (elevated in the blood most commonly by liver or bone disorders) and blood urea nitrogen (BUN) values (elevated in liver and kidney disorders) were within normal limits in all groups. Alanine aminotransferase (ALT) values (elevated with high specificity in liver disorders) were within normal limits in the saline and Dual-NPs groups, while mice in the free PTX+EVER group showed ALT levels higher than the upper limit. This increase in ALT levels, however, was not statistically significant relative to the saline and the Dual-NP groups . TherefoTo evaluate the kidney function, serum creatinine (SCR) levels were also quantified. SCR values in all groups were reported to be <18 µM/L. The low levels of SCR, along with the normal levels of blood urea nitrogen (BUN), negate the presence of kidney disorders—usually associated with high SCR levels—in all groups.max, AUCs, and half-lives of the drugs were significantly higher when administered in Dual-NPs in comparison to the free drug combination. In addition, the Dual-NPs maintained the synergistic molar ratio of PTX:EVER in the plasma for up to 24 h after administration. These results indicate that the Dual-NPs retained their stability in the blood circulation, which is essential for the successful co-delivery of PTX and EVER  to the tumor site.Previously, the Dual-NPs demonstrated good stability in vitro with less than 50% PTX and EVER released after 72 h of incubation in BSA-supplemented media at 37 °C . In the w/w). We have previously found that the drug to material ratio can significantly impact the stability of NPs. Dynamic light scattering (DLS) was used to measure the size of different untargeted NPs composed of mPEG5000-b-PLGA15,800 at different drug  to material ratios  to monitor their stability for 48 h (data not shown). Formulations with high drug to material ratios of 16.1% and 15.3%, significantly aggregated forming large clusters that could be seen with the naked eye within 6 h, indicating the low stability of the NPs. With drug to material ratios of 11.7% and 8.9%, aggregation of the NPs began after 24 h, leading to an increase in hydrodynamic diameter of about 40% and 70% of the initial size, respectively. On the other hand, with a drug to material ratio of 6.5%, the NPs maintained their initial size for 48 h at room temperature and this size was also maintained when the NPs were incubated in cell culture media containing 10% FBS at 37 °C. Therefore, the untargeted NP formulation with the lowest drug loading level (6.5%) was used for the development of Dual-NPs. The conjugation of the targeting moieties to the surface of the NPs resulted in a reduction in drug loading from 6.5% to 5.6% (w/w). Other studies have confirmed our findings and demonstrated that PLGA NPs with lower drug to material ratios are often associated with a higher degree of stability [The stability of the Dual-NPs may be attributed mainly to their low drug to material ratio or drug loading level  (mPEG-b-PDLLA) and has a drug loading level of 16% (w/w). In preclinical studies conducted in mice, the plasma AUC for PTX was 30% lower with Cynviloq than with Taxol despite the higher injected dose  [The development of a polymeric NP formulation that physically encapsulates a taxane (paclitaxel or docetaxel) and is stable in vivo has proven to be challenging ,23,24. Cctively) .w/w) in the formulation limits the maximum dose of drug that can be administered . Therefore, there must be a balance between high drug loading and sufficient in vivo stability in the design of the NP formulation to allow for superior antitumor effects.Administration of PTX and EVER in the Dual-NPs resulted in only marginal improvements in tumor growth inhibition and survival in MDA-MB-231-H2N tumor-bearing mice relative to the free-drug combination. The Dual-NP formulation was compared to free PTX+EVER at chemically equivalent doses of both drugs. One may expect that a greater degree of antitumor efficacy could be achieved for the Dual-NP formulation if a higher dose had been administered. Given the good toxicity profile observed for this formulation, it is also likely that higher doses of the formulation could be safely administered. However, the low drug loading level of 5.6%  effect (passive targeting) ,31. TherPeripheral neuropathy remains a persistent distressing side effect to patients receiving conventional PTX, interfering with their daily activities and negatively affecting their quality of life. Symptoms include numbness, tingling, burning, muscles weakness and cramping. This toxicity is seen in 70–90% of patients receiving conventional PTX, and even after discontinuation of therapy, it takes several months for symptoms of neuropathy to resolve ,35. Withb-PLGA, which has a good safety profile allowed for the elimination of the small-molecule surfactant Cremophor EL, which has been associated with PTX-induced neurotoxicity [Importantly, in the present study, neurotoxicity was found to be reduced with the administration of the drugs in the Dual-NPs relative to the free-drug combination. In addition, the formulation showed superior tolerability to free PTX+EVER in terms of body weight loss. This outcome could be attributed to the prolonged retention of the drugs within Dual-NPs in the blood circulation, enabling effective exploitation of the EPR effect which leads to selective tumor accumulation and, subsequently, reduced systemic drug exposure and associated toxicities . In additoxicity .2 and 90% relative humidity. The medium was supplemented with penicillin/streptomycin solution to 1% of final volume. Based on previous assessments of HER2 and EGFR expression by radioligand binding assays, MDA-MB-231-H2N has moderate HER2 (4.5 × 105 receptors/cell) and EGFR expression (4.8 × 105 receptors/cell) [mod/EGFRmod.The human BC cell line, MDA-MB-231-H2N, was provided by Dr. Raymond Reilly . The cells were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12) (DMEM/F12) with 10% fetal bovine serum (FBS) at 37 °C in 5% COrs/cell) . Based oFour-week old, healthy, female BALB/c mice were purchased from Charles River Laboratories  for the pharmacokinetics and toxicity studies. Four-week old, female NOD/SCID mice were purchased from an in-house breeding facility for the biodistribution studies and from Charles River Laboratories for the efficacy studies. Animal studies were conducted following protocols approved by University of Toronto’s Animal Care Committee .5000-b-PLGA15,800 and COOH-PEG-b-PLGA  were dissolved in acetone. Under magnetic stirring, following complete dissolution, the organic phase was added drop-wise to deionized water. The mixture was left under vacuum overnight at RT to evaporate the organic phase. The formulation was filtered and purified by ultrafiltration (3500 g for 10 min at RT) using ultracentrifugal filter units  of 100 kDa) . The HER2-targeted trastuzumab (TmAb) and EGFR-targeted panitumumab (PmAb) Fab fragments were generated by digestion of TmAb and PmAb IgG using immobilized papain following a well-established method [PTX+EVER-loaded NPs were prepared as previously described . Brieflyd method . The purd method . Brieflyv/v) ratio and the mixture was centrifuged for 10 min at 5000 g at 4 °C to precipitate the protein content of plasma. Supernatant was collected and the drugs were extracted by liquid-liquid extraction using t-butylmethylether (TBME). The organic phase was collected and evaporated under nitrogen before resuspending in the mobile phase .Healthy BALB/c female mice were injected intravenously (i.v.) via the tail vein with either the free PTX+EVER combination or the Dual-NP formulation at a PTX-equivalent dose of 15 mg/kg and EVER-equivalent dose of 7.5 mg/kg . The free-drug combination was dissolved in a 1:1 mixture of Cremophor EL and anhydrous ethanol. The resulting yellow viscous mixture was then diluted with saline and vortexed for 20 s to obtain a clear solution of the drugs. Mice were sacrificed by cardiac puncture under anesthesia at selected time points post-administration. Blood was collected using heparinized tubes and centrifuged at 15,000 rpm for 10 min at 4 °C to isolate plasma. Plasma was stored at −80 °C prior to analysis. Acetonitrile was added to the plasma at a 2:1 (v/v) or methanol, water, trifluoroacetic acid (95:4.9:0.1 v/v) were used as mobile phases for PTX and EVER, respectively, at a flow rate of 1.0 mL/min. A diode array detector was set at wavelengths of 227 nm and 279 nm for detection of PTX and EVER, respectively . The extraction efficiencies were 86% and 81%, the limit of detection (LOD) was 0.1 µg/mL and 0.05 µg/mL , and the limit of quantification (LOQ) was 0.4 µg/mL and 0.1 µg/mL for PTX and EVER, respectively.PTX and EVER were quantified using HPLC as previously reported . In brie1/2z) was calculated by dividing 0.693 by the slope of the terminal phase (k). The AUC0–t was calculated using the linear trapezoidal rule where t is the time of the last measurable concentration. AUCt–inf was calculated by dividing the last detected concentration by the slope of the terminal phase (k). The clearance (Cl) was estimated by the equation Cl = dose/AUC0-inf, and the volume of distribution (Vd) by Vd = dose/(AUC0-t K).Non-compartmental analysis was used to calculate all pharmacokinetic parameters as previously reported . The hal6 MDA-MB-231-H2N cells suspended in a 100 μL mixture of culture medium and Matrigel (1:1 v/v) were injected subcutaneously in the right hind limb of mice. When the tumor size reached 5–8 mm in diameter, mice were randomly allocated to the treatment or control groups. MDA-MB-231-H2N tumor-bearing female NOD/SCID mice were used for the biodistribution and efficacy studies. 1.0 × 10t test, and p-values < 0.05 were considered statistically significant.NOD/SCID female mice bearing MDA-MB-231-H2N tumors were injected i.v. into the tail vein with either free PTX+EVER or Dual-NPs at a PTX-equivalent dose of 15 mg/kg and EVER-equivalent dose of 7.5 mg/kg (PTX:EVER at a molar ratio of 1:0.5). At selected time points post-administration, mice were sacrificed and tumors, spleens, livers, hearts and kidneys were collected and stored at −80 °C. Organs obtained were homogenised in 0.9% NaCl and the resulting homogenate was extracted and quantified using the methods described above for plasma. The extraction efficiencies were 80% and 76% for PTX and EVER, respectively. The LOD was 0.2 µg/mL  for both drugs, and the LOQ was 0.5 µg/mL and 0.4 µg/mL for PTX and EVER, respectively. Data were analyzed using the Student’s w) and length (l). Tumor volume was determined using the formula v  =  π/6*l*(w2), while drug toxicity was determined by monitoring changes in animal behavior and body weight. Groups were excluded from the tumor volume efficacy study when the first mouse in each group reached the ethical endpoint. Statistical significance was calculated by the two-way ANOVA  test.NOD/SCID female mice bearing MDA-MB-231-H2N tumors received either saline, free PTX+EVER, or Dual-NPs as an i.v. dose administered weekly for eight consecutive weeks at a PTX-equivalent dose of 15 mg/kg and EVER-equivalent dose of 7.5 mg/kg (PTX: EVER at a molar ratio of 1:0.5). Tumor growth was monitored by caliper measurements of tumor width . Survival data were plotted using Kaplan-Meier survival analysis, and statistical significance was determined using the log-rank test. Four-week old, healthy, female BALB/c mice were given the following weekly treatments via i.v. tail vein injection: Dual-NPs, free PTX+EVER at a PTX-equivalent dose of 8 mg/kg (low-dose study) or 15 mg/kg (high-dose study) (PTX:EVER at a molar ratio of 1:0.5). The control group received weekly injections of saline for six consecutive weeks. Mice were sacrificed two to three days after receiving the last injection and the severity of neurotoxicity was examined using light microscopy. Segments of the sciatic nerve were embedded in formalin. Sections of 4 µm in thickness were stained with haematoxylin and eosin (H & E) before examination under light microscopy by a veterinary pathologist blinded to the groups to determine the degenerative changes in the myelinated nerve fibers. p-values < 0.05 were considered statistically significant.For mice receiving the higher dose of the drugs, blood was collected into heparinized tubes for biochemistry analysis and in Ethylenediaminetetraacetic acid (EDTA) tubes for complete blood count analysis (CBC). Blood biochemical markers were measured using the VetScan VS2  Comprehensive Diagnostic Profile, while CBC analysis was conducted using the VetScan HM5 v2.2 hematology analyzer. Mice in this group were also evaluated for liver and spleen toxicity by histopathological examination of formalin-embedded sections of the liver and spleen. Data were analyzed using the Student’s t test, and While maintaining therapeutic efficacy, Dual-NPs were successful in improving the toxicity profile of PTX relative to the free-drug combination. In addition to showing preliminary evidence of reducing neurotoxicity, Dual-NPs were significantly more tolerable than the free-drug combination with regards to body weight loss when administered at the same dose. Considering the severe toxicities associated with conventional PTX (Taxol), especially at this time when the overall survival rates for BC have improved; the development of well-tolerated medicines should be prioritized alongside the search for curative measures.


The dual luciferase assay revealed that miR-99a directly targets FGFR3 by binding its 3′ UTR in breast cancer. miR-99a was strongly down-regulated in breast tumor and FGFR3 was significantly up-regulated in breast tumor. FGFR3 silencing inhibited proliferation, migration and invasion of breast cancer cells. Deep sequencing indicated that miR-99a overexpression regulates multiple signaling pathways and triggers the alteration of the whole transcriptome. We constructed correlated expression networks based on circRNA/miRNA and lncRNA/miRNA competing endogenous RNAs regulation and miRNA-mRNA interaction, which provided new insights into the regulatory mechanism of miR-99a. In conclusion, these results suggest that the miR-99a/FGFR3 axis is an important tumor regulator in breast cancer and might have potential as a therapeutic target.MicroRNAs have been implicated in acting as oncogenes or anti-oncogenes in breast cancer by regulating diverse cellular pathways. In the present study, we investigated the effects of miR-99a on cell biological processes in breast cancer. Breast cancer cells were transfected with a lentivirus that expressed miR-99a or a scramble control sequence. Functional experiments showed that miR-99a reduced breast cancer cell proliferation, invasion and migration. Tumor xenograft experiment suggested miR-99a overexpression inhibited breast cancer cell proliferation  MicroRNAs (miRNAs) are small non-coding RNA molecules containing 18–22 nucleotides that act as agents of the RNA interference pathway and can lead to silencing of their cognate target genes by targeting mRNAs for cleavage or translational repression , and theEmerging evidence indicates that some miRNAs function as oncogenes or anti-oncogenes, which has led to a new strategy for cancer diagnostics and therapeutics. The miRNA expression profiles are very different between normal and tumor tissues, and miRNA signatures are associated with specific clinicobiological features . Breast In the present study, we explored the effects of miR-99a on cellular biological processes and identified FGFR3 as one of its target genes. Moreover, we elucidated the alteration of the whole transcriptome triggered by miR-99a using next-generation sequencing technology. Our study demonstrated that miR-99a could be a potential miRNA biomarker for breast cancer.2.The human breast cancer MCF-7 and MDA-MB-231 cell lines were obtained from the American Type Culture Collection (ATCC), and grown in Dulbecco's Modified Eagle Medium (DMEM)  supplemented with 10% fetal bovine serum  and 100 units/ml penicillin/streptomycin  at 37°C in a humidified atmosphere containing 5% COFor lentiviral construction, the hsa-miR-99a precursor sequence was cloned into the GV369 vector which contains a puromycin resistance gene. pHelper 1.0 and pHelper 2.0 are lentiviral packaging plasmids. The 293T cells were co-transfected with the recombinant GV369 plasmid, pHelper 1.0 and pHelper 2.0, and the medium was collected for the purification of viruses after 48 h. Breast cancer cell transfections were carried out at an MOI (multiplicity of infection) of 20 in the presence of 10 μg/ml polybrene. The culture medium was changed 12 h post-transfection and quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine miR-99a expression after another 72 h. The stably transfected cells were selected for 2 weeks by 5 μg/ml puromycin.3 cells per well-containing 100 μl of Dulbecco's Modified Eagle Medium  supplemented with 10% fetal bovine serum . At 0, 24, 48, 72, 96, and 120 h, cell viability was measured with the CCK8 reagent according to the manufacturer's protocol. The colony formation assay was used to investigate the colony formation ability of cells. The cells (200 cells/dishes) were plated in 60 mm culture dishes and incubated for 14 days. The colonies were counted after being fixed with 100% methanol for 15 min and stained in a 0.1% crystal violet  solution for 30 min.For the Cell Counting Kit-8 (CCK8)  assay, cells were seeded in 96-well-plates  in quintuplicate at 1 × 104 cells were plated on the 24-well-top chambers  either coated with 10 μg of Matrigel per well (for invasion assays) or uncoated (for in vitro migration assays) containing 100 μl of DMEM, and the lower chambers were filled with 600 μl of DMEM with 10% fetal bovine serum as a chemoattractant. After 24 h, the cells that did not migrate or invade through the pores were removed with a cotton swab, and the cells attached to the lower surface of the membrane were fixed with 100% methanol for 15 min and stained with 0.1% crystal violet (Biosharp) solution for 30 min. For the wound healing assay, 5 x 105 cells were plated on a 6-well-plate in DMEM with 10% fetal bovine serum. After 24 h, wounds were created with a white micropipette tip and the medium was changed to DMEM with 2% fetal bovine serum. Light microscope pictures were taken at 10x magnification at 0, 12, 24, and 48 h. Wound distances were measured in three different places.For the Transwell assay, 3 × 10For target validation, the 3′ UTR of FGFR3 was PCR-amplified and cloned downstream of firefly luciferase in the pmirGLO dual-luciferase miRNA target expression plasmid. The recombinant plasmid was sequenced to verify integrity. Three point mutations were obtained by using the QuikChange II XL Site-Directed Mutagenesis Kit  according to the manufacturer's instructions. The 293T cells were co-transfected with the recombinant plasmids and miR-99a mimic or a scramble control. After 48 h, luciferase assays were performed according to the manufacturer's instructions .Total RNA was isolated from the cultured cells using the Trizol reagent  following the manufacturer's instructions. RNA was reverse transcribed into cDNA using oligo dT primers for mRNA or specific stem-loop reverse transcription primers for miRNA with a RevertAid First Strand cDNA Synthesis Kit . Then qRT-PCR was performed using the SYBR Green PCR Super Mix  and the BIO-RAD CFX96 Real-Time PCR Detection System . The expression levels of mRNAs were normalized to GAPDH as the internal control, while miRNAs were normalized to U6 snRNA, and expression levels were calculated using the 2-ΔΔCT algorithm.Firstly, a Bioanalyzer 2100  was used to check the quantity and purity of the total RNA. Then, microRNA libraries were generated using TruSeq Small RNA Sample Prep Kits . mRNA, lncRNA, and circRNA libraries were created according to the protocol of the mRNA-Seq Sample Preparation Kit , after removing the ribosomal RNA using the Epicentre Ribo-Zero Gold Kit . Finally, single-end sequencing for miRNA and paired-end sequencing for mRNA, lncRNA and circRNA were performed on the Illumina HiSeq 2500 platform and the Illumina Hiseq 4000 platform, respectively.2(Fold-Change)| ≥ 1 and p ≤ 0.05 as the threshold for significantly differential expression.Data filtering was conducted on the raw reads output from the sequencing platform using Cutadapt  and cleain situ, respectively . Tumor volumes were assessed by caliper measurements and calculated as: V = D × d2 × 0.5 , each formed tumor was measured every 5 days with calipers. The mice were euthanized 35 days after the injection, and the tumors were weighed.Five-week-old female BALB/c nude mice were purchased from Huafukang . All animal procedures in this study were approved by the Animal Ethics Committee of Wuhan Myhalic Biotechnology Co., Ltd., and performed based on the “Guidelines for Animal Care and Use of the Model Animal Research Institute.” MCF-7 cells transfected with miR-99a or the scramble control sequence were resuspended in PBS and injected into each mouse t-tests were employed, and the data are represented as means ± SEM. p < 0.05 were statistically significant (*p < 0.05 and **p < 0.01). All the experiments were executed at least in triplicate and in independent biological replicates.Statistical analysis was performed with GraphPad Prism 5.0. Two tailed unpaired http://www.ncbi.nlm.nih.gov/geo/) of the National Center for Biotechnology Information (NCBI) is an international public repository for microarray and next-generation sequencing functional genomic data sets submitted by the research community. We retrieved the microarray data set GSE26659 (http://www.kmplot.com) was used to evaluate survival analysis between miR-99a expression level and breast cancer (http://gepia.cancer-pku.cn/)  . We retrpku.cn/) . These rFirstly, we performed qRT-PCR to confirm the transfection efficiency of lentiviral-miR-99a and lentiviral control. The results indicated that the level of mature miR-99a was increased by 7.95-fold in MCF-7 cells transfected by lentiviral-miR-99a compared to control cells . The rolin vitro. The same experiments were repeated and confirmed in MDA-MB-231 breast cancer cell line, the results were shown in The ability of miR-99a to regulate migration and invasion was investigated using the Transwell assay. We observed that cells over-expressing miR-99a showed significantly reduced migration and invasion rates compared with the control-miR transfected cells . We alsoin vitro  and biological pathway enrichment analyses using FunRich   version In addition to mRNA, there were also alterations in the expression of other RNAs between the MCF-7 cells overexpressing miR-99a and the scramble-control MCF-7 cells. To connect these differentially expressed transcriptome RNAs, we constructed correlated expression networks based on the relationships of circRNA/miRNA and lncRNA/miRNA competing endogenous RNAs regulation and miRNA-mRNA interactions .in vivo, BALB/c nude mice were injected with MCF-7 cells transfected with miR-99a or the scramble control sequence. We found that miR-99a overexpression significantly inhibited tumor growth in vivo, the tumor volumes and weights of the miR-99a overexpressed group were significantly lower than that of the control group (To determine the role of miR-99a in breast cancer progression ol group .MiR-99a is located in intron 13 of the C21orf34 gene on chromosome 21q . In the By over-expressing miR-99a in breast cancer cells, we confirmed that miR-99a inhibited the proliferative, invasive and migratory activities of breast cancer cells. Analysis based on TCGA database also suggests that low expression of mir-99a predicts a poor outcome in breast cancer. Our results were supported by those of other several studies in which miR-99a was shown to reduce the aggressiveness of breast cancer , 16, 27.Previous studies revealed that mTOR, HOXA1, and IGFBP1 were targeted by miR-99a in breast cancer –16; FGFRDespite some functional experiments and the identification of target genes, the biological mechanism of miR-99a remains poorly understood. To elucidate the underlying molecular mechanisms of miR-99a in breast cancer, we applied the deep sequencing method to detect transcriptome changes triggered by miR-99a overexpression in the MCF-7 cell line. In brief, the results demonstrated that miR-99a overexpression initiated wide-ranging alterations of the transcriptome in MCF-7 cells. Among the down-regulated mRNAs, some genes with potential oncogenic functions could be found, such as FGFR3 and EXOC8, which are also putative target genes of miR-99a. As previously mentioned, FGFR3 has been implicated in various cancers through multiple signaling pathways. It has been reported that EXOC8 fosters oncogenic ras-mediated tumorigenesis . There wIt has been reported that lncRNAs and circRNAs could function as microRNA sponges and thereby regulate gene expression at a post-transcriptional level. We detected alterations in some miRNAs, lncRNAs, and circRNAs. We observed an 7.95-fold upregulation of miR-99a-5p and proved the successful construction of a stable MCF-7 cell line over-expressing miR-99a. In addition, miR-451a showed a 9.37-fold upregulation and a high expression level in our sequencing results. Over-expression of miR-451a could inhibit cell proliferation and enhance tamoxifen sensitivity in breast cancer by regulating 14-3-3ζ, estrogen receptor α, and macrophage migration inhibitory factor , 39. TheThrough our cellular functional experiments, we demonstrated that miR-99a is a tumor suppressor gene. The deep sequencing results showed a few molecules thought to be oncogenes were up-regulated, while some RNAs that may act as tumor suppressors were down-regulated. However, this inconsistency does not affect our previous speculation. We constructed a ceRNA network to connect these molecules and found that lncRNAs and circRNAs harbor miRNAs and therefore indirectly regulate the expression of mRNAs. Post-transcriptional regulation makes it possible that the expression levels of oncogenes were increased or that the expression levels of anti-oncogenes were decreased after miR-99a overexpression. We conjecture that the mechanism by which miR-99a affects non-coding RNAs may be due to the regulation of transcription factors which were influenced by miR-99a. These pioneering discoveries might give us new insights into the regulatory mechanisms of microRNA.Taken together, our findings support an important role for miR-99a in breast cancer, both as a regulator of oncogene FGFR3 and as a tumor suppressor that results in a significant decrease in breast cancer cell proliferation, invasion, and migration. We also described the transcriptome alteration caused by miR-99a, which could lay the foundation for further research concerning this molecule in breast cancer. More research is needed on miR-99a as a potential diagnostic or prognostic biomarker.The datasets generated for this study are available on request to the corresponding author.This animal study was reviewed and approved by the Animal Ethics Committee of Wuhan Myhalic Biotechnology Co., Ltd. Written informed consent was obtained from the owners for the participation of their animals in this study.XL, YS, and YT took part in research design. YS, PY, and XL conducted experiments and drafted the manuscript. JG, QZ, and YT participated in the data analysis and helped to draft the manuscript. XL and YT took part in research funding. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The DBR-LED with SPL showed 54.3% improvement of maximal external quantum efficiency (EQE) and 65.7% enhancement of optical power at 100 mA without any degeneration in electrical properties, compared with the un-etched standard LED sample. This work has paved the way for the application of electrically-pumped UV LEDs and VCSELs based on nanoporous AlGaN DBRs.A 365-nm UV LED was fabricated based on embedded nanoporous AlGaN distributed Bragg reflectors (DBR) by electrochemical etching. The porous DBR had a reflectance of 93.5% at the central wavelength of 365 nm; this is the highest value of porous AlGaN DBRs below 370 nm which has been reported so far. An innovative two-step etching method with a SiO Besides, the epitaxy of Al(Ga)N/GaN stacks is very time-consuming because a large number of periods are required to obtain a high reflectivity. Recently, photoelectronic devices based on nanoporous GaN have been reported [+-GaN layer, and thus the refractive index is lowered equivalently. While at the same time, the unintentionally-doped GaN (u-GaN) layers will remain intact, and thus the index contrast is created. The electrochemically-etched u-GaN/n+-GaN stack structures have a high refractive index contrast and perfectly matched lattice constant, and high reflectance (>99%) can be obtained in the visible band. Porous AlGaN DBRs have also been reported [III-nitrides-based near ultraviolet light-emitting diodes (UVA LEDs) have extensive application prospects in ink printing, photoresist curing and nail phototherapy, for they are energy-efficient and mercury-free compared with traditional mercury lamps . One of reported ,16,17,18reported ,20 recen2 sidewall protection layer (SPL) to isolate the n-type layer and active region of UV LED from the electrolytes. 365-nm UV LEDs based on nanoporous AlGaN DBRs were prepared, and the etching morphology and power enhancement mechanism were analyzed as well. Finally, DBR-LED chips were fabricated and the electroluminescence (EL) spectra, voltage-current (IV) and power-current (PI) characteristics were also analyzed. The light output power of DBR-LED were substantially enhanced without any loss of electrical properties.In this work, highly-reflective nanoporous AlGaN DBRs were fabricated by electrochemical anodic oxidation method. The reflectance spectra of nanoporous AlGaN DBRs which were etched under varying bias voltages and in different electrolytes were compared. Then, we proposed an innovative two-step etching method with a SiO4) and bicyclopentadienylmagnesium (Cp2Mg) were n-type and p-type dopants, respectively. Hydrogen and nitrogen were used as carrier gases. The u-AlGaN/n+-AlGaN stack structures consist of a 20-nm-thick GaN nucleation layer grown at 580 °C, a 3-μm-thick u-GaN buffer layer grown at 1230 °C, a 500-nm-thick n-Al5%GaN (n = 1 × 1018 cm−3) as the current spreading layer, and 15.5 pairs of u-Al9%GaN/n+-Al9%GaN stacks  grown at 1250 °C. Based on the u-AlGaN/n+-AlGaN stack structures, the 365 nm DBR UV LEDs consist of a-500 nm-thick u-Al5%GaN as current blocking layer, a 1-μm-thick n-Al4%GaN layer (n = 8 × 1018 cm−3), 15 pairs of AlInGaN multiple quantum wells , a 10-nm-thick p-Al20%GaN electron-blocking layer, a 80-nm-thick p-Al6%GaN layer, and a 20-nm-thick p-GaN cap layer altogether. In the electrochemical etching procedure of the porous AlGaN DBRs, firstly a 1-μm-thick SiO2 was deposited on the wafers. Then a series of grooves with 24 μm width and 400 μm spacing were defined by standard photolithography and inductively-coupled plasma  etching methods. These Grooves were paralleled to  and 1 mol/L potassium hydroxide (KOH) solution as the electrolytes, respectively. The u-AlGaN/n+-AlGaN stacks were electrolyzed at different bias voltages under a steady voltage source  for about 4–5 h until the etching was completed. After the electrochemical etching process was over, the SiO2 was removed by diluted hydrofluoric acid, then the wafers were cleaned with deionized water and dried by nitrogen. The optical microscope images were observed on the wafer surfaces, and the cross-sectional SEM images were observed along the etching direction  system. Trimethylgallium, trimethylaluminum, trimethylindium, and ammonia were used as gallium, aluminum, indium, and nitrogen sources, respectively. Silane  was deposited on the p-GaN, and the ITO was annealed in nitrogen atmosphere at 600 °C for 4 min for better crystalline quality and higher transmittance [−4 Ω·cm, and the transmittance was about 91.1% at 365 nm, respectively. Next, Cr/Pt/Au (5 nm/500 nm/1000 nm) metals were deposited by E-beam evaporation as the n-type and p-type electrodes. Finally, the wafers were diced into chips of 450 × 750 μm2 by laser scribing.In the process of fabricating UV LEDs, a two-step etching method was introduced with a SiOmittance . The res2 using a 325-nm He-Cd laser. The electric field distribution spectra of porous DBR and DBR-LED were simulated by finite-different time-domain (FDTD) method. The EL spectra, IV curves and PI curves were measured by an ultraviolet integrating sphere  at room temperature in continuous wave (CW) conditions. The far field distribution patterns were obtained by an angle-resolved spectrum system .The pore morphologies of the porous AlGaN DBR were characterized by scanning electron microscopy . The light reflectance spectra were measured by UV-Vis-NIR spectrophotometer . The standard reference sample we used in the reflectance spectra measurement was a wide-band dielectric mirror  from Thorlabs company , because the conventional reference sample (white board) has a little bit of absorption of UV light and will result in a higher measured reflectance. The photoluminescence (PL) spectra of UV LEDs were measured by μ-PL equipment  with a spot size of less than 5 × 5 μm+-AlGaN/un-etched u-AlGaN stacks were obtained by electrochemical (EC) etching in HNO3 solution. The n+-AlGaN layers were selectively etched in nitric acid, and the porosity rises with the increase of bias voltages. The nano-pores in n+-AlGaN layer had a shape between triangle and arch, which should be attributed to the isotropic etching of HNO3 to n-AlGaN at high applied voltages, because the crystal planes become unstable and gradually disappear when the oxidation capacity of the anode becomes so strong with the increasing bias voltages [3 interface:3+ and Al3+:+-AlGaN stacks were etched in 1 M KOH solution, the morphologies of the nano-pores become totally different from the sample etched in HNO3 solution, as shown in − to Al atoms in the N-polar AlGaN surfaces [3 was mainly controlled by the n-type doping concentration, while in KOH solution it was seriously influenced by the high corrosivity of N-polar surfaces of AlGaN, thus the periodicity of porous AlGaN stacks was severely damaged. Apparently, if we want to obtain a highly reflective nanoporous AlGaN DBR by EC etching, HNO3 solution should be a better choice of the electrolyte than KOH solution, for a terrific periodicity of AlGaN stacks could be obtained by the former.voltages . The elevoltages ,23. It isurfaces . The typ9%GaN is about 2.6 at 365 nm [+-AlGaN layer could be calculated by the volume average theory (VAT) [As is known, a distributed Bragg reflector (DBR) consists of a number of quarter-wave high refractive layers and low refractive layers which are piled up periodically. The refractive index and thickness of each layer satisfy the equation of:t 365 nm , while try (VAT) ,26:(4)ne voltage ,27. In t3 under 15 V bias voltage. This is the highest reflectance achieved by porous AlGaN DBRs below 370 nm so far. It should be noted that only 15.5 pairs of u-Al/n+-Al stacks were used. Nevertheless, the porous AlGaN DBR by EC etching could reach a relatively high reflectance which must be obtained by a number of periods  in conventional AlGaN/GaN DBRs grown in situ [3), the reflectance spectrum shifted to the short wavelength side, due to the decreasing refractive index of porous n+-AlGaN layers  and thus the absorption from u-AlGaN/n+-AlGaN stacks cannot be ignored any more. Besides, the DBR etched in KOH solution had the narrowest bandwidth and the lowest reflectance, because the periodicity of u-AlGaN/n+-AlGaN stacks was severely destroyed by the etching of OH− to u-AlGaN as shown in 3-15 V) was directly imported as the simulation structure. A plane wave with a wavelength of 365 nm was set as the light source, and the boundary conditions were set as “Periodic” and “Perfect Matching Layers” for the horizontal and vertical direction, respectively. Standing waves were formed above the DBR surface which indicated the good reflection of the quarter-wave porous u-AlGaN/n-AlGaN stacks. The electric field intensity mainly focused on the top layers of AlGaN stacks, which also indicated that the number of u-AlGaN/n-AlGaN pairs could be further reduced. These results suggested that a highly-reflective porous AlGaN DBR in the UV range could be obtained by carefully adjusting the thickness and doping concentration of the un-doped/Si-doped AlGaN layers. The central wavelength of the spectrum could be easily shifted (to a certain extent) just by changing the bias voltages. Compared with the conventional in-situ Al(Ga)N/GaN DBRs, the DBRs fabricated by EC etching could easily reach a high reflectance (>93% in our work), while nearly having no lattice mismatch and the pairs required to obtain the same reflectance were also dramatically reduced due to the large index contrast. Thus, EC etching is a very promising way to replace conventional in-situ epitaxy of Al(Ga)N/GaN in fabricating highly-reflective DBRs.The reflectance spectra of porous AlGaN DBRs are shown in  in situ ,13. When2 sidewall protection layer, and the details are shown in +-Al stacks were grown in MOCVD and the wafers were cleaned before the experiment. Second, photolithography followed by ICP etching were performed to define the grooves (not mesa) with 24 μm width and 400 μm spacing, and the etching depth was 1.5 μm until the u-AlGaN was exposed. The wafers used here were split into quarters of 2 inches for easier operation of EC etching. It should be noted that the sidewalls were designed to have a sloping angle for better adhesion of SiO2, as shown in 2 was deposited by a plasma-enhanced chemical vapor deposition  system on the wafer surface, and now the sidewalls of UV LEDs were covered by a compact SiO2 layer, as shown in 2; second, ICP etching was performed successively to expose the sidewalls of AlGaN DBRs, as shown in 3 solution to obtain porous AlGaN DBRs. One should be aware that the EC etching direction was perpendicular to the grooves, because the wafer surfaces were well covered by a thick SiO2 layer, and thus the vertical etching from p-GaN surface could not happen. When the EC etching was over, the SiO2 was removed and the wafers were cleaned by deionized water and dried by nitrogen.Now that the high reflective porous AlGaN DBRs were achieved, another problem was how to combine the UV LEDs with the DBR structure together. One way to realize the composite LED structure was performing secondary epitaxy on the nanoporous AlGaN DBRs. However, the nano-pores would transform and merge with each other under a high growth temperature . So the +-AlGaN/u-AlGaN stacks were acquired, but also the n-AlGaN and active region of UV LED were still intact after EC etching, as shown in 2 SPL, which typically had a sloping angle of 38.6° as shown in 2. The SiO2 layer would fall off and SPL would be invalid if it was deposited on a vertical sidewall. The etching trenches were along the [2 SPL played an important role in the EC process.The cross-sectional SEM images of DBR-LEDs are shown in λ = 365 nm) in the in-plane direction. The dipole was put in the MQWs region of UV LED, and the refractive index of the UV LED structure was fixed at 2.60 to simplify the simulation. In the ST-LED, the electric field had a uniform distribution inside and only a small amount of photons escaped from the LED surface because of the total internal reflection. In the DBR-LED, the electric field intensity was enhanced in the UV-LED region and reduced in the DBR region, due to the high reflection of bottom porous DBR. The amount of emitting photons were also dramatically increased, which indicated the DBR LED would have a higher light extraction efficiency than the ST-LED.Finally, DBR-LED chips were fabricated by standard LED chip technology after EC etching. The EL spectra of DBR-LED and ST-LED chips are shown in The optical and electrical test results are demonstrated in 2 SPL was proposed to protect the n-AlGaN and active region of UV LEDs from the electrolyte during EC etching. The cross-sectional morphologies of DBR-LED with/without SPL were compared. PL spectra of DBR-LEDs were measured and the resonance effect was observed. FDTD simulations were performed to analyze the electric field distribution of DBR-LED and ST-LED, and the results indicated that the light extraction efficiency of UV LED was enhanced due to the highly-reflective porous DBR. The electrical and optical properties of DBR-LED chips were also analyzed. The EL emission spectra were measured, and the EL intensity of DBR-LED was enhanced compared with the ST-LED. In the IV measurement, the DBR-LED without SPL encountered an increasing operating voltage and series resistance due to the porosification of n-AlGaN layer of UV LED, while the voltage of DBR-LED with SPL remained the same as the un-etched ST-LED. In the PI measurement, the maximal EQE of UV LED was improved by 54.3%, and the output power was enhanced by 65.7% at 100 mA by the use of nanoporous AlGaN DBR. Comprehensively, DBR-LED with SPL showed substantial enhancement of light output power, without any degeneration in electrical properties. In addition, the DBR-LED also had a smaller divergence angle due to the angle-dependent reflectivity of porous AlGaN DBR, and this is more beneficial for application such as UV curing. We believe that by optimizing the thickness and Al mole fraction of the u-AlGaN current-blocking layer and n-AlGaN layer of UV LED, the absorption would be reduced and the output power would be further enhanced. This work has paved the way for the application of electrically-pumped UV LEDs and VCSELs based on nanoporous AlGaN DBRs.In summary, we have fabricated nanoporous AlGaN DBRs with the electrochemical etching method. The etching mechanisms were analyzed and the electric field distribution of nanoporous AlGaN stacks was also simulated by the FDTD method. A peak reflectance of 93.5% at the central wavelength of 365 nm was achieved and it was the highest reflectance below 370 nm of porous AlGaN DBRs which has been reported so far. In order to fabricate the DBR-LED, an innovative two-step etching method with SiO